Infections with sexually transmitted pathogens, including Chlamydia trachomatis, increase the transmission of HIV-1 across the female genital tract. We will study the mechanisms by which C. trachomatis specifically increases HIV-1 transmissibility across the human endocervix, the primary site of entry for this combination of pathogens. We will build upon our polarizable in vitro models for endocervical epithelia and preliminary data showing a decrease in epithelial integrity and an increase in endocervical HIV-1 receptor and co-receptor expression in the presence of C. trachomatis infection. We propose to investigate the effects of C. trachomatis serovar D on three potential pathways for epithelial transmission of HIV-1, including: 1) epithelial cell entry, integration and productive infection, 2) epithelial cell entry and transcytosis from the apical to basal surface of the endocervical epithelia and 3) paracellular transport of HIV-1 virions between epithelial cells. Within these investigations, we will define detailed mechanisms for C. trachomatis-associated increases in the endocervical cell surface expression the HIV-1 receptors GalCer, CXCR4 and CCR5. We will investigate the roles of endocervical cell tight junction components (catenin, cadherin, nectin and afadin) in our documented decrease in endocervical integrity upon infection with C. trachomatis. In the absence of vaccines against C. trachomatis and HIV-1, effective vaginal microbicides may be our best approach to the prevention of heterosexual spread of each of these sexually transmitted infections. The in vitro models developed for this project will serve as a platform for future rational phase 1 testing of novel components of vaginal microbicides against C. trachomatis and HIV-1, including selective estrogen receptor modulators (SERMs), toll-like receptor modulators and probiotics. Our in vitro models for dual infection with C. trachomatis and HIV-1 will be one of the first to allow the study of interactions between sexually transmitted pathogens. The role of co-infection and superinfection with other sexually-transmitted pathogens in HIV transmission is understudied. Our dual infection models can begin to fill this gap and can act as templates for the development of similar dual-infection models in other reproductive mucosal epithelial sites, including rectal mucosa, vaginal mucosa and ectocervical mucosa. PUBLIC HEALTH RELEVANCE: Infection with the common sexual pathogen, Chlamydia trachomatis, increases the heterosexual transmission of HIV-1. Using a novel in vitro model of the human endocervical epithelium, the site primarily implicated in C. trachomatis/HIV interactions, we will study the mechanisms behind the effects of C. trachomatis on HIV-1 binding to the endocervix, entry into the endocervix, integration into the genome of the epithelial cell, epithelial cell transcytosis and paracellular transport across the endocervical epithelium. The findings in this R21 will provide a platform for future R01 funding to rationally test vaginal microbicidal components against dual pathogen infections and their local toxicities.